Project Summary/Abstract Background. The current proposal is a competitive renewal application that focuses on defining the mechanisms causing zonation of the adrenal cortex. In the mammalian adrenal cortex, mineralocorticoids are produced in the zona glomerulosa (ZG) and glucocorticoids in the zona fasciculata (ZF). Production of aldosterone results from ZG-specific expression of the enzyme aldosterone synthase (Cyp11b2), while production of glucocorticoids relies on ZF-specific expression of the closely related 11?-hydroxylase (Cyp11b1). In normal physiology, the aldosterone production in the ZG is tightly regulated by the renin-angiotensin II system. However, dysregulation of zonation with renin-independent aldosterone production and Cyp11b2 expression occurs in primary aldosteronism (PA), a condition that affects 8% of hypertensive patients. The current proposal takes advantage of our past molecular research on Cyp11b2 and moves our findings into in vivo models of mammalian zonation and adrenal disease. Research goals. Specific Aim 1 will define the contribution of Dax1 (Nr0b1) in normal adrenocortical zonation. Hypothesis for Aim 1: Dax1 expression is required for adrenal zone-specific Cyp11b2 and Cyp11b1 expression. Approach for Aim 1: Cell and mouse based approaches will be used to define the interplay between Sf1 and Dax1 in the regulation of adrenal cell production of aldosterone (ZG phenotype) vs. corticosterone (ZF phenotype). Primary cultures of mouse adrenal cells and the H295R human adrenocortical cell line will be used to define the molecular mechanisms by which Sf1 and Dax1 cooperatively regulate gene expression that defines ZG versus ZF steroid production. Mouse genetic models will be used to interrogate the sexual dimorphic roles of Dax1 in adrenal zonation and corresponding ZG Cyp11b2 versus ZF Cyp11b1 expression. Specific Aim 2 will define mechanisms responsible for sex differences in adrenal aldosterone production and susceptibility to PA. Hypothesis for Aim 2: The differential expression of the nuclear receptor Dax1 in female vs male adrenals directly impacts Cyp11b2 expression, aldosterone production, and susceptibility to PA. Rationale for Aim 2: Four mouse models of PA have shown greater disease susceptibility in females compared to males. In addition, women have a higher prevalence of low renin hypertension and a higher aldosterone/renin ratio compared to men. The molecular mechanisms controlling these differences are not known. Physiological manipulations and transgenic mouse experiments are proposed to test hypotheses associated with both project aims. Innovations. There are at least three key innovative components to the proposed research: 1) This will represent the first development of an inducible mouse model of PA; 2) First research designed to define the role for sexual dimorphism in adrenal zonation and PA; 3) First to apply broad based LC-MS/MS determination of steroids (30 steroids) to define the steroid metabolome of male and female mice. Significance: The proposed studies will provide a detailed understanding of the molecular mechanisms responsible for adrenal functional zonation (ZG vs ZF) and the mechanisms leading to PA.